Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture

Monolayer cell cultures and cartilage tissue fragments have been used to examine the effects of hydrostatic fluid pressure (HFP) on the anabolic and catabolic functions of chondrocytes. In this study, bovine articular chondrocytes (bACs) were grown in porous three-dimensional (3-D) collagen sponges, to which constant or cyclic (0.015 Hz) HFP was applied at 2.8 MPa for up to 15 days. The effects of HFP were evaluated histologically, immunohistochemically, and by quantitative biochemical measures. Metachromatic matrix accumulated around the cells within the collagen sponges during the culture period. There was intense intracellular, pericellular, and extracellular immunoreactivity for collagen type II throughout the sponges in all groups. The incorporation of [(35)S]-sulfate into glycosaminoglycans (GAGs) was 1.3-fold greater with constant HFP and 1.4-fold greater with cyclic HFP than in the control at day 5 (P < 0.05). At day 15, the accumulation of sulfated-GAG was 3.1-fold greater with constant HFP and 2.7-fold with cyclic HFP than the control (0.01). Quantitative immunochemical analysis of the matrix showed significantly greater accumulation of chondroitin 4-sulfate proteoglycan (C 4-S PG), keratan sulfate proteoglycan (KS PG), and chondroitin proteoglycan (chondroitin PG) than the control (P < 0.01). With this novel HFP culture system, 2.8 MPa HFP stimulated synthesis of cartilage-specific matrix components in chondrocytes cultured in porous 3-D collagen sponges.     

In vitro induction of cartilage-specific macromolecules by a bone extract

An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.     

SOMITE CHONDROGENESIS : A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54–60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse serum (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utilized to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 Å) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200–800 Å protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many cells die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitic mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most non-induced cultures fed medium containing FCS and F12X There is more cell death, less matrix accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites.     

Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery

Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor beta1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and beta1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components

Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly.     

Ethanol Exposure Stimulates Cartilage Differentiation by Embryonic Limb Mesenchyme Cells

Studies of neural, hepatic, and other cells have demonstrated thatin vitroethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformitiesin vivo,prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23–25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant ofin vitrochondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in³⁵SO₄incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol onin vitrochondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.     

Properties of chick embryo chondrocytes grown in serum-free medium

Chick embryo tibial chondrocyte growth and activities were compared in serum-free and serum-supplemented media. A basal salts medium containing equal volumes of Ham's F-12 and Dulbecco's modified Eagle's medium was supplemented with 10% fetal calf serum or with a mixture of bovine insulin, transferrin, fibroblast growth factor, dexamethasone, a prostaglandin E1 supplement, and a liposome supplement. Chondrocytes grew at identical rates in both media. Insulin, liposomes, and fibroblast growth factor were required for optimum growth in the serum-free medium, but removal of transferrin, dexamethasone, or prostaglandin E1 had little effect on the growth rate. In the serum-supplemented medium, the chondrocytes synthesized Type II collagen, Mr = 59,000 collagen, and both the large, cartilage-specific and the small ubiquitous proteochondroitin SO4 species typically produced by cultured chondrocytes. In the serum-free medium there was a shift toward synthesis of Type I collagen and a loss of the capacity to synthesize Mr = 59,000 collagen and the cartilage-specific proteochondroitin SO4. The loss of capacity for cartilage-specific proteochondroitin SO4 synthesis began immediately after replacement of the serum with the mixture of defined growth factors and the rate of loss was retarded but not reversed when serum was added back in place of the growth factors. When the serum and the mixture of growth factors were added together to the basal medium at the time of cell plating, the chondrocytes grew rapidly and retained their normal phenotype observed in serum-supplemented cultures. Thus, the serum appears to contain factors which are required for retention of the chondrocyte phenotype in culture over and above those factors necessary for cell growth.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes

Excessive mechanical load is thought to be responsible for the onset of osteoarthrosis (OA), but the mechanisms of cartilage destruction caused by mechanical loads remain unknown. In this study we applied a high magnitude cyclic tensile load to cultured chondrocytes using a Flexercell strain unit, which produces a change in cell morphology from a polygonal to spindle-like shape, and examined the protein level of cartilage matrixes and the gene expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and proinflammatory cytokines such as IL-1beta and TNF-alpha. Toluidine blue staining, type II collagen immunostaining, and an assay of the incorporation of [35S]sulfate into proteoglycans revealed a decrease in the level of cartilage-specific matrixes in chondrocyte cultures subjected to high magnitude cyclic tensile load. PCR-Southern blot analysis showed that the high magnitude cyclic tensile load increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1beta, TNF-alpha and TIMP-1 in the cultured chondrocytes, while the mRNA level of MMP-2 and TIMP-2 was unchanged. Moreover, the induction of MMP-1, MMP-3 and MMP-9 mRNA expression was observed in the presence of cycloheximide, an inhibitor of protein synthesis. These findings suggest that excessive mechanical load directly changes the metabolism of cartilage by reducing the matrix components and causing a quantitative imbalance between MMPs and TIMPs.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro

Tissue engineering of articular cartilage from chondrocytes or stem cells is considered to be a potential aspect in the treatment of cartilage defects. In order to optimize culture conditions the influence of low oxygen tension (5%) - single or in combination with intermittent hydrostatic pressure (HP: 30/2 min on/off loading; 0.2 MPa) - on the biosynthetic activity (sulfate and proline incorporation) of human osteoarthritic chondrocytes cultured on collagen I/III membranes was investigated. Additionally, chondrogenesis from high density or monolayer cultures of bovine adherent bone marrow cells (aBMC) with and without chondrogenic medium supplements (CM) was analyzed by RT-PCR (mRNA expression of aggrecan and collagen type II). We could show that low oxygen tension increases significantly the biosynthesis of collagen I/III membrane-associated chondrocytes and even higher under co-stimulation with HP. While there is no chondrogenesis in monolayer cultures, CM induces expression of cartilage matrix molecules in high density cultures of aBMC which is even increased under the influence of low oxygen tension. Both, low oxygen tension and HP without CM are alone not sufficient stimuli for chondrogenesis. It can be concluded that low oxygen tension and HP might be useful tools in cartilage tissue engineering and that these physico-chemical factors promote but do not induce chondrogenesis under the given conditions.     

Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen ( approximately 5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose-derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two-fold increase in the rate of protein synthesis and a three-fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage-associated matrix molecules, including collagen type II and chondroitin-4-sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue-engineered cartilage.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study

Mesenchymal stem cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, which suggest these cells as an attractive cell source for cartilage tissue engineering approaches. Our objective was to study the effects of TGF-beta1, hyaluronic acid and synovial fluid on chondrogenic differentiation of equine MSC. For that, bone marrow was aspirated from the tibia of one 18-month-old horse (Haflinger) and MSC were isolated using percoll-density centrifugation. To promote chondrogenesis, MSC were centrifuged to form a micromass and were cultured in a medium containing 10 ng/ml TGF-beta1 or 0.1mg/ml hyaluronic acid (Hylartil, Ostenil) or either 5%, 10% or 50% autologous synovial fluid as the chondrogenesis inducing factor. Differentiation along the chondrogenic lineage was documented by type II collagen and proteoglycan expression. MSC induced by TGF-beta1 alone showed the highest proteoglycan expression. Combining TGF-beta1 with hyaluronic acid could not increase the proteoglycan expression. Cultures stimulated by autologous synovial fluid (independent of concentration) and hyaluronic acid demonstrated a pronounced, but lower proteoglycan expression than cultures stimulated by TGF-beta1. The expression of cartilage-specific type II collagen was high and about the same in all stimulated cultures. In summary, hyaluronic acid and autologous synovial fluid induces chondrogenesis of equine mesenchymal stem cells, which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chondrogenic differentiation.     

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast-like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures. Isolated mesenchymal cells from 12-day-old mouse limb buds were grown in alginate for up to 4 weeks. A sub-population of the cells differentiated to chondrocytes and exhibited a stable phenotype until the end of the culture period. After 3 to 4 days a cartilage-specific matrix started to develop. Fibroblast-like cells from this mixed culture did not survive; they became necrotic. When alginate was later on dissolved by chelating agents, only chondrocytes were isolated. During dissolution of alginate and centrifugation, chondrocytes did not lose their contact with their new matrix present on their surfaces. Cultivation of these chondrocytes or chondrones in mass culture yields a pure chondrocyte population. Immunoelectron microscopic investigations revealed collagen type II, fibronectin, decorin and chondroitin sulfate-proteoglycans in the chondrocyte capsules and in mass culture.